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Development of Anti-Isoproturon Polyclonal Antibody
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Agricultural Sciences in China 2007, 6(8): 964-969
fL ScienceDirect
*
August 2007
Development of Anti-Isoproturon Polyclonal Antibody LI Fang-shil, SUN Fengl, LIU Xian-jin2 and CUI Heng-huaz I
Departmen! of Applied Chemistry, College of Science, Nanjing University of Technology, Nanjing 210009, P.R. China Food Safety Research and Inspection Center, Jiangsu Academy of Agriculrural Sciences, Nanjing 210014, P.R.China
Abstract A competitive enzyme-linked immunosorbent assay (ELISA) suitable for the determination of the urea herbicide isoproturon, 3-(4-isopropylphenyl)-1.1-dimethylurea, in food and environmental samples was developed. Two haptens named 143carboxypropyl)-3-(4-isopropylphenyl)-1-methylurea (hapten 4C) and 1-(5-carboxypentyl)-3-(4-isopropylphenyl)-1methylurea (hapten 6C) were synthesized. The haptens were coupled to bovine serum albumin (BSA) and ovalbumin (OVA), respectively, using the N-hydroxysuccinimide reaction. The hapten 6C-BSA conjugate was used as the immunogen, with which a high-titer anti-isoproturon polyclonal antibody (pAb) was successfully obtained by immunization of New Zealand white rabbits. The hapten 4C-OVA conjugate was used as coating antigen and a method of the indirect competitive ELISA for isoproturon was established. The haptens were confirmed with TLC, IR. and LH NMR. The conjugation molar ratios of hapten 4C to OVA and hapten 6C to BSA were 36: 1 and 46: 1, respectively, as calculated by a UV spectrophotometry. The highest titer of the anti-isoproturon sera determined by a non-competitive indirect ELISA procedure was 1.6 x 105. The optimal concentrations of the coating antigen and the dilution of the anti-isoproturon sera used in the ELISA were 0.1 mg L-1 and 1.Ox 105, respectively. The concentration of isoproturon that inhibits 50% of antibody-antigen binding (IC,) was 0.07 mg mL-1. The cross-reactivities of six urea herbicides including chlorbromuron, fluometuron, monolinuron were lower than 0.1%. Isoproturon is a small molecule without immune activity and active functional group for attaching to carrier protein. To produce an antibody against isoproturon with high titer and high specificity is the most important step in the development of an immunochemical method for the determination of isoproturon in food and environmental samples. The two haptens synthesized in this study have carboxyl groups and accommodate different lengths of spacer arms, and the phenyl and isopropyl groups are fully exposed. An anti-isoproturon polyclonal antibody with high titer and high specificity was successfully obtained by immunization of rabbits with the conjugate of the hapten attached to the protein carrier.
Key words: isoproturon, hapten, artificial antigen, polyclonal antibody, enzyme-linked immunosorbent assay (ELISA)
INTRODUCTION Isoproturon, 3-(44sopropylphenyl)- 1,l -dimethylurea, is the most common representative of the urea herbicides. It is mainly used for controllingannual grasses and many annual broad-leaved weeds (Su 1989; Terry 1998). Since it was used heavily for years, it has contaminated
soil, water, and other natural environment. At present, methodologies reported for the detection of urea herbicides, such as isoproturon, mainly include gas chromatography (GC) and high-performance liquid chromatography (HPLC). While GC is preferred in pesticide residue analysis, the urea herbicides are not directly amenable to GC, because of their thermal instability. The GC methods often lack adequate
This paper is translated from its Chinese version in Scientia Agriculrura Sinica. LI Fang-shi, Ph D, Professor, TeVFax: +86-25-83587450. E-mailfangshi.li@njut,edu.cn
Development of Anti-Isoproturon Polyclonal Antibody
sensitivity and require complex sample preparation [cumbersome clean-up, preliminary enrichment, and derivatization procedures (Huang et al. 2003)l. HPLC methods often need clean-up steps, such as solid-phase extraction (Li et al. 2001). They cannot be used to screen great numbers of samples. It is very urgent and important to develop a simple and fast method to monitor isoproturon residues at very low levels in environment. Recently, immunochemical methods, including enzyme-linked immunosorbent assay (ELISA), have been used for herbicide analyses. They are fast, simple, sensitive, and especially suitable to fast screen largescale samples and for in site detection. Immunoassay for isoproturon has been reported overseas (Mouvet et al. 1997; Mallat et al. 2001). Isoproturon is a small molecular. It has neither immune activity nor active group for connecting to carrier protein. The key factor to develop an immunoassay for isoproturon residues is to prepare anti-isoproturon antibody. Katmeh et al. (1994) and Ben Rejeb et al. (1998) described the methods for preparation of antiisoproturon sheep antibody and anti-isoproturon rabbit antibody, respectively. In this study, the methods for the preparation of two isoproturon haptens with different lengths of spacer arms and of anti-isoproturon antibody with high titer were developed.
965
MATERIALS AND METHODS
3,3’, 5,5’-tetramethylbenzidine (TMB, Merker), N, N’dime-thylfomanide (DMF, Sigma, USA), dialysis cassettes (MW 14 OOOD, Huamei, Shanghai, China), goat anti-rabbit conjugated to HRP (IgG-HRP, Baiao Bioinforbody, Zhuhai, China). All other regents were of analytical grade and were purchased from Sinopham, Shanghai, China. Mobile phase for TLC (V,,,, acetate:Vn-hexane:Vaetic acid = 49:49:2, prepared before use). Buffers and solutions Coating buffer (CBS, 0.05 mol L-’ carbonate buffer, pH 9.6), phosphate-buffered saline (PBS, 0.15 mol L-’ phosphate buffer, pH 7.4), PBST [PBS with 0.05% (v/v) Tween-201, citrate-phosphate buffer (CPBS, 0.1 mol L-I citric acid monohydrate and 0.2 mol L-I Na,HPO4-12H,O,pH 5.5), substrate solution (50 pL of 0.65% H,O, added to 19.75 mL of citratephosphate buffer containing 10 mg mL-’ TMB), and stop solution (2 mol L-I H,SO,). New Zealand white rabbits were bred by Jiangsu Academy of Agricultural Sciences, China. Instruments NMR spectrometer (AV-300, Brucker, Switzerland), IR spectrometer (Nexus-670, Nicolet, USA), UV-Vis spectrometer (Lambda-25, Perkin Elmer, USA), 78-1 magnetic force mix appearance (Hangzhou Electromotor, China), DG503 1 microplate reader and DG3080 washing machine (Huadong Electronics, China), 96 well microtiter plates (Corning, USA), pipettors (Jencons Sealpette, USA), ultrapure water system (Labconco, USA), analytical balance (Ohaus, USA).
Materialsand instruments
Test methods
Materials Standards of isoproturon and other urea herbicides (398%, Fluka). Stock solutions were prepared in ethanol with the concentration of 1 mg mL-’ and were stored at 4°C. N-methylpyrrolidone (CP, Guanghua Technical, Shanghai, China), Nmethylcaprolactam ( 399%, Sanhe Chemical, Hangzhou, China), 4-isopropylphenylisocyanate(20% w/w in xylene, Kuaida-Agrochem, Rudong, China), Nhydroxysuccinimide (NHS, 3 97%), N, N’dicyclohexylcarbodiimide (DCC, 3 99%, Merker, USA), complete and incomplete Freund’s adjuvants (FCA and FICA, Merker), bovine serum albumin (BSA, MW 67 OOO, Merker), ovalbumin (OVA, MW 45 O00, Merker),
Hapten synthesis Isopruton is a small molecular, it does not have immuno-activityby itself, and any suitable functional group to be conjugated with carrier proteins. Two isopruton haptens with carboxylic groups were synthesized (Fig. 1). The coating hapten, 1-(3carboxypropyl)-3-(4-isopropylphenyl)-1-methylurea (hapten 4C) was prepared f r o m 4-methylaminobutanoic acid (Benson and Cairns 1948) and 4isopropylphenylisocyanate. The immunogen hapten, 1-(5-carboxypenyl)-3-(4-isopropylphenyl)- 1methylurea (hapten 6C), was prepared from 6methylaminohexanoic acid (Karu et al. 1994) and 4isopropylphenylisocyanate. The purity of each hapten
(02007.CAAS.All rights W W I V ~ ~Published . by ElseMr Ltd.
LI Fang-shi
966
ef
al.
A Hapten 4C
x
A Hapten 6C
Fig. 1 Reaction scheme of the synthesis of isoproturon haptens.
was confirmed by thin-layer chromatography (TLC), 'H-NMR, and infrared (IR) spectra. Preparation of hapten-protein conjugates The hapten 6C was conjugated to BSA and the hapten 4C to OVA, respectively, by the activated ester method. For that, 0.2 mmol of hapten 6C or hapten 4C was dissolved in 1 mL of anhydrous DMF. While stirring, 0.2 mmol of NHS and 0.2 mmol of DCC were added. The reaction mixture was continuously stirred for 4 h at room temperature and then for 12 h at 4°C. The reaction mixture was centrifuged (6 min, 1400 r/min) to remove the urea precipitate. The clear supernatant (800 mL) was slowly added to BSA or OVA solution and the solution was stirred for 16 h at 4°C. The dialysis was carried out with 0.2 mol L-'PBS (0.9% (w/v) NaCl) for 4 d. The conjugations were scanned by UV-Vis spectrometry to confirm the conjugation. Solutions were then freeze-dried and stored at -20°C. Production of antibody The method of raising polyclonal antibody was according to Dong et al. (2001) and Xu (1991). Three New Zealand white rabbits were immunized with the hapten 6C-BSA conjugate. For primary injection, 1 mg of protein conjugate was dissolved in 1 mL of sterile physiological saline and emulsified with an equal volume of Freund's adjuvant. About 1 mL of the emulsion was intradermally injected at different sites on the back of each rabbit. After three weeks, booster injections with the use of Freund's incomplete adjuvant were administered in the same
manner fortnightly. The dosage was doubled for the last booster. Since the second booster, the rabbits were bled through the ear vein at the eighth day after each booster injection to monitor the antibody production. The final bleeding was done through the heart. For separation of cells, the blood was left at 4°C overnight. The serum was decanted and then centrifuged at 4 000 rimin for 10 min. The sera were collected and purified by saturated (NH,),SO,. Conveniently sized portions were stored at -20°C. Characterization of the antisera (1) The titers of the antisera The antiserum titers were determined by the checker-board titration using an indirect ELISA procedure (Ma et a l . 1998) performed as follows. The microtiter plates were coated with 100 &/well of coating antigen (hapten 4C-OVA conjugate) of different concentration in carbonate buffer (0.05 mol L-l, pH 9.6) and were allowed to stand overnight at 4°C. The plates were washed three times with PBST using the automatic plate washer and thoroughly tapped dry. Sites not occupied with the coating antigen were blocked for 1 h at 37°C with 200 &/well 1.O% OVA in PBS buffer. After washing the plates, 100 pL of antiserum series diluted with PBS was added to each well and incubated for 2 h at 37°C. The plates were washed again and 100 pL of goat anti-rabbit IgG conjugated to horseradish peroxidase diluted 5 OOO times with PBS was added to each well and incubated for 1 h
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Development of Anti-Isoproturon Polyclonal Antibody
at 37°C. After washing, 100 pL of the substrate solution (TMB) was added to each well. Finally, the enzyme reaction was stopped after 15 min by the addition of 50 pL of 2 mol L-I sulfuric acid. The absorbance was measured at 450 nm. The dilution times of antiserum and the concentration of coating antigen yielding a response of OD 1.O at 450 nm were used as the working concentration. (2) Affinity and specificity of the pAb Six urea herbicides, hlorbromuron, fluometuron, monolinuron, tebuthiuron, thidiazuron, and siduron, were used to test the cross-reactivity. Under the optimal concentration of the coating antigen and the antiserum, a dilution series of each compound in methanol was determined. The determination method was similar as the titer test. Microcal origin soft (Marshall 1995) was used to draw the inhibition curves. The IC, values (molar concentration of inhibitor that produces a 50% decrease of the maximum normalized response) of the isoproturon and its analogues were obtained from the inhibition curves. The inhibition ratio and the cross reactivity were calculated based on the following equation (Abad et al. 1999): Inhibition ratio (%) = [(OD- - ODh) - (OD,- ODm)] x 1OO/( OD,= - OD,,). OD- and OD,, are the absorbance with and without herbicide, respectively. ODxis the absorbance of the blank. Cross reactivity (%) = (IC50of isoproturon/IC,, of analogue) x 100%.
967
Hz, CH2-2), 1.72 (2H, CH,-3), 1.17 (d, 6H, 2CH,).
Identificationof hepten 6C The structure of hapten 6C was confirmed by TLC, IR, and 'HNMR. TLC: R,=0.33 (INz4). IR (KBr): 3 378 cm-' (s, NH),1715 cm-' (vs, COOH), 1623 cm-' (vs, amide C = 0),1525 cm-' (vs, amide H), 1243 cm-' (m, C-0). 'HNMR (DMSO-d,): 612.0 (lH, OH), 8.08 (s, IH, NH),7.32 (d, J=8.1 Hz, 2H, ArH-a, b), 7.05 (d, J=8.1 Hz, 2H, ArH-C,d), 3.25 (t, J = 7.2 Hz, 2H, CH,-6), 2.89 (s, 3H, NCH,), 2.79 (heptet, lH, CH), 2.18 (t, J=7.2 Hz, CH,-2), 1.5 (m, 4H, CH,-3, 5 ) , 1.25 (m, 2H, CH,4), 1.15 (d, J=6.8 Hz, 6H, 2 CH,).
Hapten-proteinconjugatesand coupling ratio The UV spectra of the solutions of the haptens, proteins, and the conjugates were shown in Figs.2 and 3. Compared with hapten 4C and OVA, the UV spectrum of hapten 4C-OVA was different. At the 1- of hapten 4C (240 nm), the absorbance of hapten 4C was larger than that of OVA. Based on the principle of the addition of absorbance, the hapten 4C-OVA conjugate was confirmed. The hapten 6C-BSA was confirmed in the same way. The coupling ratios of hapten 6c to BSA and hapten 4C to OVA were calculated with the following equation (Liu et aZ. 2000) as 46:l and 36:1, respectively.
RESULTS
ratio = ('240
conjugation
- '240
carrier protd''24~l
haptcn
identification of hepten 4C The structure of hapten 4C was confirmed by TLC, IR, and 'HNMR. TLC: Rf=0.23 (UV,,). IR (KBr): 3 345 cm-I (s, NH),1715 cm-' (vs, COOH), 1 657 cm-' (vs, amide C = 0),1 536 cm-' (vs, amide H), 1231 cm-' (m, C-0). 'H-NMR(DMS0-d,): 612.1 (lH, OH), 8.1 (s, lH, NH), 7.34 (d, J=8.3 Hz, 2H, ArH-2,6), 7.08 (d, J=8.3 Hz,2H, ArH-3,5), 3.29 (t, J=7.2 Hz, 2H, CH,-4), 2.91 (s, 3H, NCH,), 2.81 (heptet, lH, CH), 2.22 (t, J=7.2
200
250
300
3.50
400
Wavelength (nm)
Fig. 2 UV spectra of hapten 4C, OVA, and hapten 4C-OVA.
92007. CAAS. All Whhi reserved. PuMLshedby EkevierLM.
LI Fang-shi et al.
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Tters of the antisera
showed that the cross-reactivities of the herbicides were all less than 0.1%. The antibody had high specificity.
Through the immune program, all of the three rabbits yielded high titer antibodies. When the concentration of the coating antigen was 1.0 mg mL-l, the titers of the antisera were 1.6 x lo5, 2.0 x lo4, and 8.0 x lo4for the A4746, A5205, and A505 1 rabbit, respectively. The A4746 antiserum had the highest titer and was used in the following study. The optimal concentration and the dilution times of the competitive indirect ELISA were 0.1 mg mL-' and 1.Ox lo5, respectively.
The specificity of the antibody Fig.4 is the inhibition curve of isoproturon by competitive indirect ELISA. The IC,, was 0.07 mg d - I . The values of IC, of hlorbromuron, fluometuron, monolinuron, tebuthiuron, thidiazuron,and siduron were also got in the same way. The results of the calculation 3.6
-
e,
g
2 '
w n 4
1 -
0.0
200
I
250
300
350
400
DISCUSSION The key factor to develop an immunoassay for the detection of pesticide residues in agricultural and environmental samples is to prepare a specific antibody. Because most pesticides ke small molecules, they have no immune activity themselves. The preparation of haptens is often difficult. The herbicide isoproturon in itself cannot be used as an immunogen because of its low molecular weight (MW206). In this study, 4isopropylphenylisocyanate, N-methylcaprolactam, and N-methylpyrrolidone were used as reactants. The two haptens synthesized have carboxyl groups with different lengths of spacer arms, and the phenyl and isopropyl groups are fully exposed. An anti-isoproturonpolyclonal antibody with high titer and high specificity was successfully obtained by immunization of rabbits with the conjugate of the hapten attached to the carrier protein. The highest titer of the anti-isoproturon sera determined by a non-competitive indirect ELISA procedure was 1.6 x 10'. The cross-reactivities of six urea herbicides were less than 0.1%. The produced antibody could fulfiil the determination of isoproturon resides by ELISA. Using the produced antibody, an ELISA method has been established to detect isoproturon residues in water, crops, and soil samples (Zhang and Li 2005). The ELISA results were consistent with the HPLC results.
Wavelength (nm)
Fig. 3 UV spectra of hapten 6C, BSA, and hapten 6C-BSA.
S=3.30794414 .E c c
r = 0.09687057 34.40
17.20 0.00 I 1 .o
10.0
100.0
1 000.0
10 000.0
Concentration of isoproturon (W m L ')
Fig. 4 Standard inhibition curve of antigen-antibody reaction.
CONCLUSION The key factor of determination of chemical pesticides by immunoassay is to prepare antibodies with high titer and high specificity. Isoproturon itself has neither immune activity nor active group for conjugation to carrier protein. The two mimics (hapten 6C and hapten 4C) prepared in this study have carboxyl groups and accommodate different lengths of spacer arms, and the phenyl and isopropyl groups are fully exposed. They possess the characteristics of isoproturon haptens. The haptens were confirmed by TLC, IR, and " M R . Antigens were produced by conjugating hapten 6C
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reserved.PuMishedby ElsevierUd.
Development of Anti-Isoproturon Polyclonal Antibody
to BSA and hapten 4C to OVA, respectively, with the active ester method. The coupling ratios of hapten 6C to BSA and hapten 4C to OVA were determined as 46: 1 and 36: 1, respectively. An anti-isoproturon antibody with high titer and high specificity was successfully obtained by immunization of rabbits with the hapten 6C-BSA conjugate. The ELISA titer of the antiserum was 1.6 x lo5. The crossreactivities of the six urea herbicides with similar structures as isoproturon were less than 0.1%. Based on the inhibition curve of isoproturon, the IC,, was 0.07 mg mL-I. The produced anti-isoproturon antibody could fulfill the determination of isoproturon residues in environmental and agricultural samples.
Acknowledgements This work was financially supported by National Natural Science Foundation of China (20447003).
References Abad A, Moreno M J, Montoya A. 1999. Development of monoclonal antibody based immunoassays to the Nmethylcarbamate pesticide carbofuran. Journal ofAgricultural and Food Chemistry, 47,2475-2485. Ben Rejeb S, Fischer-Durand N, Martel A, Le Goffic F, Lawrence J F, Yeung J M, Abbott M A. 1998. Development and validation of an indirect enzyme immunoassay for the determination of the herbicide isoproturon in water matrices. International Journal of Environmental Analytical Chemistry, 69, 13-30, Benson R E, Cairns T L. 1948. Chemical reactions of caprolactam. Journal of American Chemical Society, 70,2115-2118. Dong G W, Wang M, Liu X J, Yu X Y.2001. The production of polyclone antibody for indirect competitive ELISA for methamidophos residue. Journal of Huazhong Agricultural University, 20, 340-343. (in Chinese) Huang C, Gan P S, Yang Y. 2003. Determination of isoproturon in rice using derivatization and gas chromatography. Chinese
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Journal of Health Laboratory Technology, 13, 7 10-711. (in Chinese) Karu A E, Goodrow M H, Schmidt D J, Hammock B D, Bigelow M W. 1994. Synthesis of haptens and derivation of monoclonal antibodies for immunoassay of the phenylurea herbicide diuron. Journal ofAgricultura1and Food Chemistry, 42, 301-309. Katmeh M F, Frost G, Aherne W, Stevenson D. 1994. Development of an enzyme-linked immunosorbent assay for isoproturon in water. Analyst, 119,431-435. Li F S, Martens D, Kettrup A. 2001. Simultaneous determination of sixteen phenylurea herbicides in water by high performance liquid chromatography and solid phase extraction. Chinese Journal of Chromatography, 19, 534-531. Liu S Z, Feng D H, Chen M J, Qian C F. 2000. Study on a highly specific enzyme-linked immunosorbent assay for carbofuran. J o u m l of Analytical Science, 16,373-378. (in Chinese) Ma Y,Liu H. 1998. Immunology Tutorialfor Graduate Students. Dalian Press, Dalian. (in Chinese) Mallat E, Barzen C, Abuknesha R, Gauglitz G, Barcelo D. 2001. Part per trillion level determination of isoproturon in certified and estuarine water samples with a direct optical immunosensor. Analytica Chimica Acta, 426,209-2 16. Marshall J C. 1995. The solution for technical graphics. Analytical Chemistry, 67, A424-A425. Mouvet C, Broussard S, Riolland H, Baran N, Abuknesha R, Ismail G. 1997. Evaluation of ELISA microtiter plate-based assays for the direct determination of isoproturon in water samples and soil extracts. Chemosphere, 35, 1099-1 116. Su S Q. 1989. Conspectus of Herbicides. Science Press, Beijing. pp. 182-183. (in Chinese) Terry R R. 1998. Metabolic Pathways of Agrochemicals. The Royal Society of Chemistry, Cambridge. pp. 735-739. Xu Y W . 1991. Immunoanalytical Techniques. 2nd ed. Science Press, Beijing. pp. 393-394. (in Chinese) Zhang J, Li F S. 2005. Determination of isoproturon and its degradation product 4-isopropylaniline in soil by highperformance liquid chromatography. Journal of AgroEnvironment Science, 24, 1000-1002. (in Chinese) (Edited by WANG Lu-han)
Agricultural Sciences in China 2007, 6(8): 964-969
fL ScienceDirect
*
August 2007
Development of Anti-Isoproturon Polyclonal Antibody LI Fang-shil, SUN Fengl, LIU Xian-jin2 and CUI Heng-huaz I
Departmen! of Applied Chemistry, College of Science, Nanjing University of Technology, Nanjing 210009, P.R. China Food Safety Research and Inspection Center, Jiangsu Academy of Agriculrural Sciences, Nanjing 210014, P.R.China
Abstract A competitive enzyme-linked immunosorbent assay (ELISA) suitable for the determination of the urea herbicide isoproturon, 3-(4-isopropylphenyl)-1.1-dimethylurea, in food and environmental samples was developed. Two haptens named 143carboxypropyl)-3-(4-isopropylphenyl)-1-methylurea (hapten 4C) and 1-(5-carboxypentyl)-3-(4-isopropylphenyl)-1methylurea (hapten 6C) were synthesized. The haptens were coupled to bovine serum albumin (BSA) and ovalbumin (OVA), respectively, using the N-hydroxysuccinimide reaction. The hapten 6C-BSA conjugate was used as the immunogen, with which a high-titer anti-isoproturon polyclonal antibody (pAb) was successfully obtained by immunization of New Zealand white rabbits. The hapten 4C-OVA conjugate was used as coating antigen and a method of the indirect competitive ELISA for isoproturon was established. The haptens were confirmed with TLC, IR. and LH NMR. The conjugation molar ratios of hapten 4C to OVA and hapten 6C to BSA were 36: 1 and 46: 1, respectively, as calculated by a UV spectrophotometry. The highest titer of the anti-isoproturon sera determined by a non-competitive indirect ELISA procedure was 1.6 x 105. The optimal concentrations of the coating antigen and the dilution of the anti-isoproturon sera used in the ELISA were 0.1 mg L-1 and 1.Ox 105, respectively. The concentration of isoproturon that inhibits 50% of antibody-antigen binding (IC,) was 0.07 mg mL-1. The cross-reactivities of six urea herbicides including chlorbromuron, fluometuron, monolinuron were lower than 0.1%. Isoproturon is a small molecule without immune activity and active functional group for attaching to carrier protein. To produce an antibody against isoproturon with high titer and high specificity is the most important step in the development of an immunochemical method for the determination of isoproturon in food and environmental samples. The two haptens synthesized in this study have carboxyl groups and accommodate different lengths of spacer arms, and the phenyl and isopropyl groups are fully exposed. An anti-isoproturon polyclonal antibody with high titer and high specificity was successfully obtained by immunization of rabbits with the conjugate of the hapten attached to the protein carrier.
Key words: isoproturon, hapten, artificial antigen, polyclonal antibody, enzyme-linked immunosorbent assay (ELISA)
INTRODUCTION Isoproturon, 3-(44sopropylphenyl)- 1,l -dimethylurea, is the most common representative of the urea herbicides. It is mainly used for controllingannual grasses and many annual broad-leaved weeds (Su 1989; Terry 1998). Since it was used heavily for years, it has contaminated
soil, water, and other natural environment. At present, methodologies reported for the detection of urea herbicides, such as isoproturon, mainly include gas chromatography (GC) and high-performance liquid chromatography (HPLC). While GC is preferred in pesticide residue analysis, the urea herbicides are not directly amenable to GC, because of their thermal instability. The GC methods often lack adequate
This paper is translated from its Chinese version in Scientia Agriculrura Sinica. LI Fang-shi, Ph D, Professor, TeVFax: +86-25-83587450. E-mailfangshi.li@njut,edu.cn
Development of Anti-Isoproturon Polyclonal Antibody
sensitivity and require complex sample preparation [cumbersome clean-up, preliminary enrichment, and derivatization procedures (Huang et al. 2003)l. HPLC methods often need clean-up steps, such as solid-phase extraction (Li et al. 2001). They cannot be used to screen great numbers of samples. It is very urgent and important to develop a simple and fast method to monitor isoproturon residues at very low levels in environment. Recently, immunochemical methods, including enzyme-linked immunosorbent assay (ELISA), have been used for herbicide analyses. They are fast, simple, sensitive, and especially suitable to fast screen largescale samples and for in site detection. Immunoassay for isoproturon has been reported overseas (Mouvet et al. 1997; Mallat et al. 2001). Isoproturon is a small molecular. It has neither immune activity nor active group for connecting to carrier protein. The key factor to develop an immunoassay for isoproturon residues is to prepare anti-isoproturon antibody. Katmeh et al. (1994) and Ben Rejeb et al. (1998) described the methods for preparation of antiisoproturon sheep antibody and anti-isoproturon rabbit antibody, respectively. In this study, the methods for the preparation of two isoproturon haptens with different lengths of spacer arms and of anti-isoproturon antibody with high titer were developed.
965
MATERIALS AND METHODS
3,3’, 5,5’-tetramethylbenzidine (TMB, Merker), N, N’dime-thylfomanide (DMF, Sigma, USA), dialysis cassettes (MW 14 OOOD, Huamei, Shanghai, China), goat anti-rabbit conjugated to HRP (IgG-HRP, Baiao Bioinforbody, Zhuhai, China). All other regents were of analytical grade and were purchased from Sinopham, Shanghai, China. Mobile phase for TLC (V,,,, acetate:Vn-hexane:Vaetic acid = 49:49:2, prepared before use). Buffers and solutions Coating buffer (CBS, 0.05 mol L-’ carbonate buffer, pH 9.6), phosphate-buffered saline (PBS, 0.15 mol L-’ phosphate buffer, pH 7.4), PBST [PBS with 0.05% (v/v) Tween-201, citrate-phosphate buffer (CPBS, 0.1 mol L-I citric acid monohydrate and 0.2 mol L-I Na,HPO4-12H,O,pH 5.5), substrate solution (50 pL of 0.65% H,O, added to 19.75 mL of citratephosphate buffer containing 10 mg mL-’ TMB), and stop solution (2 mol L-I H,SO,). New Zealand white rabbits were bred by Jiangsu Academy of Agricultural Sciences, China. Instruments NMR spectrometer (AV-300, Brucker, Switzerland), IR spectrometer (Nexus-670, Nicolet, USA), UV-Vis spectrometer (Lambda-25, Perkin Elmer, USA), 78-1 magnetic force mix appearance (Hangzhou Electromotor, China), DG503 1 microplate reader and DG3080 washing machine (Huadong Electronics, China), 96 well microtiter plates (Corning, USA), pipettors (Jencons Sealpette, USA), ultrapure water system (Labconco, USA), analytical balance (Ohaus, USA).
Materialsand instruments
Test methods
Materials Standards of isoproturon and other urea herbicides (398%, Fluka). Stock solutions were prepared in ethanol with the concentration of 1 mg mL-’ and were stored at 4°C. N-methylpyrrolidone (CP, Guanghua Technical, Shanghai, China), Nmethylcaprolactam ( 399%, Sanhe Chemical, Hangzhou, China), 4-isopropylphenylisocyanate(20% w/w in xylene, Kuaida-Agrochem, Rudong, China), Nhydroxysuccinimide (NHS, 3 97%), N, N’dicyclohexylcarbodiimide (DCC, 3 99%, Merker, USA), complete and incomplete Freund’s adjuvants (FCA and FICA, Merker), bovine serum albumin (BSA, MW 67 OOO, Merker), ovalbumin (OVA, MW 45 O00, Merker),
Hapten synthesis Isopruton is a small molecular, it does not have immuno-activityby itself, and any suitable functional group to be conjugated with carrier proteins. Two isopruton haptens with carboxylic groups were synthesized (Fig. 1). The coating hapten, 1-(3carboxypropyl)-3-(4-isopropylphenyl)-1-methylurea (hapten 4C) was prepared f r o m 4-methylaminobutanoic acid (Benson and Cairns 1948) and 4isopropylphenylisocyanate. The immunogen hapten, 1-(5-carboxypenyl)-3-(4-isopropylphenyl)- 1methylurea (hapten 6C), was prepared from 6methylaminohexanoic acid (Karu et al. 1994) and 4isopropylphenylisocyanate. The purity of each hapten
(02007.CAAS.All rights W W I V ~ ~Published . by ElseMr Ltd.
LI Fang-shi
966
ef
al.
A Hapten 4C
x
A Hapten 6C
Fig. 1 Reaction scheme of the synthesis of isoproturon haptens.
was confirmed by thin-layer chromatography (TLC), 'H-NMR, and infrared (IR) spectra. Preparation of hapten-protein conjugates The hapten 6C was conjugated to BSA and the hapten 4C to OVA, respectively, by the activated ester method. For that, 0.2 mmol of hapten 6C or hapten 4C was dissolved in 1 mL of anhydrous DMF. While stirring, 0.2 mmol of NHS and 0.2 mmol of DCC were added. The reaction mixture was continuously stirred for 4 h at room temperature and then for 12 h at 4°C. The reaction mixture was centrifuged (6 min, 1400 r/min) to remove the urea precipitate. The clear supernatant (800 mL) was slowly added to BSA or OVA solution and the solution was stirred for 16 h at 4°C. The dialysis was carried out with 0.2 mol L-'PBS (0.9% (w/v) NaCl) for 4 d. The conjugations were scanned by UV-Vis spectrometry to confirm the conjugation. Solutions were then freeze-dried and stored at -20°C. Production of antibody The method of raising polyclonal antibody was according to Dong et al. (2001) and Xu (1991). Three New Zealand white rabbits were immunized with the hapten 6C-BSA conjugate. For primary injection, 1 mg of protein conjugate was dissolved in 1 mL of sterile physiological saline and emulsified with an equal volume of Freund's adjuvant. About 1 mL of the emulsion was intradermally injected at different sites on the back of each rabbit. After three weeks, booster injections with the use of Freund's incomplete adjuvant were administered in the same
manner fortnightly. The dosage was doubled for the last booster. Since the second booster, the rabbits were bled through the ear vein at the eighth day after each booster injection to monitor the antibody production. The final bleeding was done through the heart. For separation of cells, the blood was left at 4°C overnight. The serum was decanted and then centrifuged at 4 000 rimin for 10 min. The sera were collected and purified by saturated (NH,),SO,. Conveniently sized portions were stored at -20°C. Characterization of the antisera (1) The titers of the antisera The antiserum titers were determined by the checker-board titration using an indirect ELISA procedure (Ma et a l . 1998) performed as follows. The microtiter plates were coated with 100 &/well of coating antigen (hapten 4C-OVA conjugate) of different concentration in carbonate buffer (0.05 mol L-l, pH 9.6) and were allowed to stand overnight at 4°C. The plates were washed three times with PBST using the automatic plate washer and thoroughly tapped dry. Sites not occupied with the coating antigen were blocked for 1 h at 37°C with 200 &/well 1.O% OVA in PBS buffer. After washing the plates, 100 pL of antiserum series diluted with PBS was added to each well and incubated for 2 h at 37°C. The plates were washed again and 100 pL of goat anti-rabbit IgG conjugated to horseradish peroxidase diluted 5 OOO times with PBS was added to each well and incubated for 1 h
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Development of Anti-Isoproturon Polyclonal Antibody
at 37°C. After washing, 100 pL of the substrate solution (TMB) was added to each well. Finally, the enzyme reaction was stopped after 15 min by the addition of 50 pL of 2 mol L-I sulfuric acid. The absorbance was measured at 450 nm. The dilution times of antiserum and the concentration of coating antigen yielding a response of OD 1.O at 450 nm were used as the working concentration. (2) Affinity and specificity of the pAb Six urea herbicides, hlorbromuron, fluometuron, monolinuron, tebuthiuron, thidiazuron, and siduron, were used to test the cross-reactivity. Under the optimal concentration of the coating antigen and the antiserum, a dilution series of each compound in methanol was determined. The determination method was similar as the titer test. Microcal origin soft (Marshall 1995) was used to draw the inhibition curves. The IC, values (molar concentration of inhibitor that produces a 50% decrease of the maximum normalized response) of the isoproturon and its analogues were obtained from the inhibition curves. The inhibition ratio and the cross reactivity were calculated based on the following equation (Abad et al. 1999): Inhibition ratio (%) = [(OD- - ODh) - (OD,- ODm)] x 1OO/( OD,= - OD,,). OD- and OD,, are the absorbance with and without herbicide, respectively. ODxis the absorbance of the blank. Cross reactivity (%) = (IC50of isoproturon/IC,, of analogue) x 100%.
967
Hz, CH2-2), 1.72 (2H, CH,-3), 1.17 (d, 6H, 2CH,).
Identificationof hepten 6C The structure of hapten 6C was confirmed by TLC, IR, and 'HNMR. TLC: R,=0.33 (INz4). IR (KBr): 3 378 cm-' (s, NH),1715 cm-' (vs, COOH), 1623 cm-' (vs, amide C = 0),1525 cm-' (vs, amide H), 1243 cm-' (m, C-0). 'HNMR (DMSO-d,): 612.0 (lH, OH), 8.08 (s, IH, NH),7.32 (d, J=8.1 Hz, 2H, ArH-a, b), 7.05 (d, J=8.1 Hz, 2H, ArH-C,d), 3.25 (t, J = 7.2 Hz, 2H, CH,-6), 2.89 (s, 3H, NCH,), 2.79 (heptet, lH, CH), 2.18 (t, J=7.2 Hz, CH,-2), 1.5 (m, 4H, CH,-3, 5 ) , 1.25 (m, 2H, CH,4), 1.15 (d, J=6.8 Hz, 6H, 2 CH,).
Hapten-proteinconjugatesand coupling ratio The UV spectra of the solutions of the haptens, proteins, and the conjugates were shown in Figs.2 and 3. Compared with hapten 4C and OVA, the UV spectrum of hapten 4C-OVA was different. At the 1- of hapten 4C (240 nm), the absorbance of hapten 4C was larger than that of OVA. Based on the principle of the addition of absorbance, the hapten 4C-OVA conjugate was confirmed. The hapten 6C-BSA was confirmed in the same way. The coupling ratios of hapten 6c to BSA and hapten 4C to OVA were calculated with the following equation (Liu et aZ. 2000) as 46:l and 36:1, respectively.
RESULTS
ratio = ('240
conjugation
- '240
carrier protd''24~l
haptcn
identification of hepten 4C The structure of hapten 4C was confirmed by TLC, IR, and 'HNMR. TLC: Rf=0.23 (UV,,). IR (KBr): 3 345 cm-I (s, NH),1715 cm-' (vs, COOH), 1 657 cm-' (vs, amide C = 0),1 536 cm-' (vs, amide H), 1231 cm-' (m, C-0). 'H-NMR(DMS0-d,): 612.1 (lH, OH), 8.1 (s, lH, NH), 7.34 (d, J=8.3 Hz, 2H, ArH-2,6), 7.08 (d, J=8.3 Hz,2H, ArH-3,5), 3.29 (t, J=7.2 Hz, 2H, CH,-4), 2.91 (s, 3H, NCH,), 2.81 (heptet, lH, CH), 2.22 (t, J=7.2
200
250
300
3.50
400
Wavelength (nm)
Fig. 2 UV spectra of hapten 4C, OVA, and hapten 4C-OVA.
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LI Fang-shi et al.
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Tters of the antisera
showed that the cross-reactivities of the herbicides were all less than 0.1%. The antibody had high specificity.
Through the immune program, all of the three rabbits yielded high titer antibodies. When the concentration of the coating antigen was 1.0 mg mL-l, the titers of the antisera were 1.6 x lo5, 2.0 x lo4, and 8.0 x lo4for the A4746, A5205, and A505 1 rabbit, respectively. The A4746 antiserum had the highest titer and was used in the following study. The optimal concentration and the dilution times of the competitive indirect ELISA were 0.1 mg mL-' and 1.Ox lo5, respectively.
The specificity of the antibody Fig.4 is the inhibition curve of isoproturon by competitive indirect ELISA. The IC,, was 0.07 mg d - I . The values of IC, of hlorbromuron, fluometuron, monolinuron, tebuthiuron, thidiazuron,and siduron were also got in the same way. The results of the calculation 3.6
-
e,
g
2 '
w n 4
1 -
0.0
200
I
250
300
350
400
DISCUSSION The key factor to develop an immunoassay for the detection of pesticide residues in agricultural and environmental samples is to prepare a specific antibody. Because most pesticides ke small molecules, they have no immune activity themselves. The preparation of haptens is often difficult. The herbicide isoproturon in itself cannot be used as an immunogen because of its low molecular weight (MW206). In this study, 4isopropylphenylisocyanate, N-methylcaprolactam, and N-methylpyrrolidone were used as reactants. The two haptens synthesized have carboxyl groups with different lengths of spacer arms, and the phenyl and isopropyl groups are fully exposed. An anti-isoproturonpolyclonal antibody with high titer and high specificity was successfully obtained by immunization of rabbits with the conjugate of the hapten attached to the carrier protein. The highest titer of the anti-isoproturon sera determined by a non-competitive indirect ELISA procedure was 1.6 x 10'. The cross-reactivities of six urea herbicides were less than 0.1%. The produced antibody could fulfiil the determination of isoproturon resides by ELISA. Using the produced antibody, an ELISA method has been established to detect isoproturon residues in water, crops, and soil samples (Zhang and Li 2005). The ELISA results were consistent with the HPLC results.
Wavelength (nm)
Fig. 3 UV spectra of hapten 6C, BSA, and hapten 6C-BSA.
S=3.30794414 .E c c
r = 0.09687057 34.40
17.20 0.00 I 1 .o
10.0
100.0
1 000.0
10 000.0
Concentration of isoproturon (W m L ')
Fig. 4 Standard inhibition curve of antigen-antibody reaction.
CONCLUSION The key factor of determination of chemical pesticides by immunoassay is to prepare antibodies with high titer and high specificity. Isoproturon itself has neither immune activity nor active group for conjugation to carrier protein. The two mimics (hapten 6C and hapten 4C) prepared in this study have carboxyl groups and accommodate different lengths of spacer arms, and the phenyl and isopropyl groups are fully exposed. They possess the characteristics of isoproturon haptens. The haptens were confirmed by TLC, IR, and " M R . Antigens were produced by conjugating hapten 6C
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Development of Anti-Isoproturon Polyclonal Antibody
to BSA and hapten 4C to OVA, respectively, with the active ester method. The coupling ratios of hapten 6C to BSA and hapten 4C to OVA were determined as 46: 1 and 36: 1, respectively. An anti-isoproturon antibody with high titer and high specificity was successfully obtained by immunization of rabbits with the hapten 6C-BSA conjugate. The ELISA titer of the antiserum was 1.6 x lo5. The crossreactivities of the six urea herbicides with similar structures as isoproturon were less than 0.1%. Based on the inhibition curve of isoproturon, the IC,, was 0.07 mg mL-I. The produced anti-isoproturon antibody could fulfill the determination of isoproturon residues in environmental and agricultural samples.
Acknowledgements This work was financially supported by National Natural Science Foundation of China (20447003).
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